Evaluation of different methods detecting intracellular generation of free radicals

Abstract

Reactive oxygen species (ROS) play several biological roles. We investigated the applicability of fluorescent probes for their detection (i) in rabbit lens epithelial cells during ageing in culture, and (ii) in thin sections of rat heart. We used dihydroethidium (DHE), dichlorofluorescin (DCFH), and dihydrorhodamine 123 (DHR) together with detection of autofluorescence both in cells and in chloroform extracts. Superoxide production was confirmed by a specific histochemical method using Mn(2+). All methods demonstrated higher production of ROS in older cells. All probes revealed different sites of ROS production in young and old cells and could be used for investigation of ROS generation during cell ageing. In the thin sections of rat heart DCFH was not suitable for intracellular ROS detection. The results indicate that the potential of fluorescent dyes in ROS detection is not usually fully exploited, and that blue autofluorescence is associated with oxidative damage.

Protein damage and death by radiation in Escherichia coli and Deinococcus radiodurans

“…Levels of PC in both D. radiodurans and E. coli increased as a function of dose in parallel with the generation of ROS monitored by oxidation of the intracellular dye 2,4-dihydrorhodamine (DHR) to a form that fluoresces at 530 nm (8) (Fig. 1 C and D).…”

AMP-Activated Protein Kinase Inhibits Homocysteine-Induced Dysfunction and Apoptosis in Endothelial Progenitor Cells

“…At higher levels, superoxide anions will react with NO to form a cytotoxic peroxynitrite [9,10,26]. We use H 2 DCF-DA to detect ROS accumulation because it can interact with most reactive species stimulated by Hcy [27,28]. Here we found that AICAR (0.5 and 1 mmol/L) markedly suppressed Hcy (500 μmol/L) induced ROS accumulation in EPCs.…”

Selenoprotein H Suppresses Cellular Senescence through Genome Maintenance and Redox Regulation

“…Images were captured under a light microscope for quantification. Autofluorescence is another index of aging in cultured human diploid fibroblasts (40). Passage 4 SelH and scrambled shRNA MRC-5 cells were subjected to Zeiss AxioObserver 100 fluorescence microscope at 200ϫ magnification.…”